The efficacy of hormone replacement therapy (HRT) in preventing postmenopausal bone loss is well-known. However, the relative roles of the two known estrogen receptors (ERs), alpha and beta, and their bone-specific mechanisms of action continue to be debated. For example, biochemical studies outlined below agree that ERalpha has profound inhibitory effects on osteoclast-mediated bone resorption by tonically down-regulating osteoblast cytokine production. Yet, recently reported 'double knock-out' mouse animal models for both ERalpha and ERbeta so far have shown no osteoporotic phenotype, despite clear effects of estrogen (E2)-deficiency in the uterus and gonads. The inability to reconcile data from in vitro cell culture experiments and the mouse double ERalpha/beta knockout studies have made it difficult to formulate a hypothesis regarding ER mechanism of action in bone. This is especially true since the established animal model for post-menopausal osteoporosis (OP)-the rat- is not yet accessible to genetic manipulation by transgenic and knock-out technologies. Thus, while the rat model can continue to test the efficacy of new therapies, it cannot be adapted to functionally characterize the molecular mechanisms of ERalpha and ERbeta action and their role in OP. In an effort to shed light on ERalpha and ERbeta function in bone, re report implementation of a mouse model of ovariectomy (OVX)-induced OP as a system for further elucidating the molecular mechanisms in hormone-dependent bone homeostasis. As a 'proof of principle' experiment for testing this model system, we propose to introduce characterized, constitutively action mutant ER alpha isoform (CAM-ERalpha) as transgenes into our mice. These two ERalpha isoforms, L536P and Y537S encode adjacent single residue changes in the ERalpha ligand-binding domain, and have been previously shown to confer full gene activation to ERalpha in the absence of E2 using in vitro cell-based assays. We will confirm the activity the CAM-ERalpha (L536P) and (Y537S) on MC-3T3-E1 osteoblastic cells. We then will determine whether these receptors can prevent bone loss in the absence of HRT in vivo by expressing CAM-ERalpha transgenes under the transcriptional control of an inducible 'Tet On/Off promoter system designed for regulating mammalian gene expression. If this pilot transgenic study demonstrates CAM-ERalpha isoforms can rescue the OP phenotype from OVX mice in the absence of HRT, it will be a key initial experiment in: 1) elucidating the molecular mechanisms of ERalpha in bone homeostasis in an in vivo assay, and 2) developing a mouse model system for OVX-induced OP that can be manipulated to add, delete, or alter specific gene targets that are active in bone.